Thrombin is postulated to mediate neointimal vascular lesion formation at sites of denuding vascular injury. This hypothesis has been tested in baboons by measuring the effects of hirudin, a potent direct antithrombin, on the formation of vascular lesions induced by implanting arteriovenous vascular grafts (AVGs). Hirudin was delivered locally into the blood flow boundary layer by retroviral-transduced hirudin-secreting vascular endothelial cells (ECs) covering AVG luminal surfaces. Measurements of neointimal lesion formation at distal graft-vascular anastomoses were compared for AVG-ECs that had been transduced with a retroviral construct containing a cDNA encoding hirudin vs non-hirudin control ECs. Stable hirudin-secreting transduced-ECs were established in cell culture. Antithrombotic efficacy of EC-secreted hirudin was demonstrated for thrombogenic segments interposed in chronic exteriorized femoral arteriovenous (AV) shunts. AVG flow surfaces were endothelialized by a ttaching hirudin-transduced vs non-hirudin control ECs at confluent density in vitro. EC-lined AVGs were surgically implanted in baboons as bilateral brachial AVGs, one covered with non-hirudin control ECs, and the contralateral AVG covered with hirudin-secreting ECs. Hirudin-secreting ECs decreased thrombus formation on segments of collagen-coated graft interposed in chronic AV shunts. Hirudin-secreting ECs also reduced platelet deposition in propagated thrombotic tails extending downstream from segments of vascular graft. Thus, hirudin-transduced ECs produced levels of hirudin locally that decreased thrombin receptor- (TR)-dependent platelet deposition without compromising hemostasis systemically. ECs covering luminal flow surfaces of implanted AVGs remained attached throughout the 30-day period, as shown by initial imaging studies of attached 111In-EC following AVG implantation, subsequent scanning electron microscopy, and by recovery of hirudin secreting ECs from 30-d AVG implan ts that generated 17"7 (range 9-25) ng/106cells/24 hr hirudin. AVGs covered with hirudin-secreting ECs reduced neointimal lesion formation at distal graft-vessel anastomoses, i.e., 1.02 " 0.14 mm2 vs 1.82 " 0.15 mm2 in contralateral AVGs bearing non-hirudin control ECs (p <0.01). As anticipated, ipsilateral upstream arterial-AVG anastomotic lesion formation was not significantly decreased, i.e., 1.26 " 0.92 mm2 vs 1.99 " 0.86 mm2 (p>0.1). Viral vector-directed secretion of hirudin from ECs lining implanted AVGs significantly reduces neotintimal vascular lesion formation, indicating the importance of thrombin in the production of neointimal proliferative lesions.